REVIEW ARTICLE

published: 02 June 2014

doi: 10.3389/fnmol.2014.00050

Systemic gene delivery to the central nervous system using

Adeno-associated virus
Mathieu Bourdenx 1,2 , Nathalie Dutheil 1,2 , Erwan Bezard 1,2 and Benjamin Dehay 1,2 *
1
Institut des Maladies Neurodgnratives, UMR 5293, Universit de Bordeaux, Bordeaux, France
2
CNRS, Institut des Maladies Neurodgnratives, UMR 5293, Bordeaux, France

Edited by: Adeno-associated virus (AAV)-mediated gene delivery has emerged as an effective and safe
Deniz Dalkara, Universit Pierre et tool for both preclinical and clinical studies of neurological disorders. The recent discovery
Marie Curie, France
that several serotypes are able to cross the bloodbrain barrier when administered
Reviewed by:
Alessandro Vercelli, University of
systemically has been a real breakthrough in the eld of neurodegenerative diseases.
Turin, Italy Widespread transgene expression after systemic injection could spark interest as a
Douglas M. McCarty, The Research therapeutic approach. Such strategy will avoid invasive brain surgery and allow non-focal
Institute at Nationwide Childrens gene therapy promising for CNS diseases affecting large portion of the brain. Here, we will
Hospital, USA
review the recent results achieved through different systemic routes of injection generated
*Correspondence:
Benjamin Dehay, Institut des
in the last decade using systemic AAV-mediated delivery and propose a brief assessment
Maladies Neurodgnratives, UMR of their values. In particular, we emphasize how the methods used for virus engineering
5293, Universit de Bordeaux, 146 could improve brain transduction after peripheral delivery.
rue Lo Saignat, 33076 Bordeaux
Cedex, France Keywords: gene therapy, AAV, neurological disorders, neurodegenerative diseases, systemic delivery
e-mail: benjamin.dehay@u-bordeaux.fr
Erwan Bezard and Benjamin Dehay
are senior authors.

INTRODUCTION et al., 2013). Taking advantage of progresses made in rAAV

In the last decade, adeno-associated-virus (AAV)-mediated gene
delivery has emerged as an effective and safe tool for both pre-
clinical and clinical studies of neurological disorders (Bartus
et al., 2013; Weinberg et al., 2013; Ojala et al., 2014). Currently,
AAV is the most widely used vector for clinical trials for neu-
rological disorders (gene therapy database can be found at:
http://www.abedia.com/wiley/index.html). To date, no adverse
effects linked to the use of this vector have ever been reported
from clinical trials. Adeno-associated virus is a non-pathogenic
dependovirus from the parvoviridae family requiring helper func-
tions from other viruses, such as adenovirus or herpes simplex
virus, to fulll its life cycle (Dayton et al., 2012). The wild-type
(WT) AAV is characterized by a single-stranded DNA (ssDNA)
genome, with inverted terminal repeats (ITR) at both ends, of
approximately 5 kb surrounded by a capsid. Advances in process
development have made AAV production fast, reliable, highly pure,
and affordable.
The rst recombinant AAVs of serotype 2 (rAAV2) have been
generated in the 1980s after the removal of 96% of the viral
genome (Samulski et al., 1982; Hermonat and Muzyczka, 1984;
McLaughlin et al., 1988). Only the two ITRs containing repli-
cation origin and encapsidation signal remained making it a
safe, non-replicative virus. Further studies allowed the produc-
tion of high-titer rAAV batches in the absence of WT virus
and adenovirus (Ferrari et al., 1997; Grimm et al., 1998; Xiao
et al., 1998). Moreover, AAVs have been reported to transduce
both dividing and non-dividing cells as well as a wide range
of tissue while remaining being poorly immunogenic, making
it an ideal candidate for gene delivery to the CNS (Weinberg
production, the rst clinical trials for neurological disorders,
such as Parkinsons disease, using rAAV2 vectors opened a
new era (Kaplitt et al., 2007; Marks et al., 2010; Bartus et al.,
2013).
Phylogenic studies of capsid protein sequence from human
and non-human primate (NHP) tissue allowed the characteri-
zation of distinct clades or families (Gao et al., 2004). Recently,
12 of these AAV serotypes have been engineered into rAAV
(Porras et al., 2014). Thanks to their different capsid composition,
the multiple serotypes exhibit distinct transduction proles com-
pared to rAAV2 (Vandenberghe et al., 2008). The ability of AAV2
ITRs to package any of the serotype capsids allows efcacy com-
parison between serotypes in vivo (Rabinowitz et al., 2002). To
date, while rAAV2 is the most widely used in clinical trials, most of
the other serotypes have shown an enhanced ability to transduce
neurons in experimental studies (Davidson et al., 2000; Taymans
et al., 2007; Tarantal and Lee, 2010).
It is difcult to dene the best serotype for intraparenchymal
CNS injections since species and cerebral structures have been
shown to inuence transduction success (Taymans et al., 2007;
Korecka et al., 2011; Weinberg et al., 2013). At least one study
reported that overexpression of the microtubule-associated pro-
tein tau peaked earlier when mediated by rAAV9 or rAAVrh10
than by rAAV2 or rAAV8 despite their 2.1-fold lower dose of virus
genome (vg; Klein et al., 2008).
Among the different serotypes, only a few have been shown
to efciently cross the bloodbrain barrier (BBB; Zhang et al.,
2011). The BBB deprives the brain of > 98% of neurotherapeu-
tic compounds (Pardridge, 2002). In this context, gene therapy

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Bourdenx et al.
has been proposed as a means of crossing the BBB (Foust et al.,
2008). Widespread transgene expression after systemic injec-
tion, although challenging, could be of interest for therapeutic
approaches. Such a strategy would avoid invasive brain surgery and
allow promising non-focal gene therapy for CNS diseases such as
lysosomal storage disorders (LSDs) or Alzheimers disease, which
knowingly affect large part of the brain.
WIDESPREAD BRAIN TRANSDUCTION AFTER SYSTEMIC
INJECTION
As this eld of research is booming, we review here the recent
results achieved through different systemic routes of injection,
such as intramyocardialy, intramuscularly, and intravascularly,
generated in the last decade using systemic AAV-mediated delivery.
In addition, we propose a brief assessment of their values.
Successful gene therapies for brain diseases require a
widespread distribution and magnitude of transgene expression
throughout the brain. Several studies reported an efcient gene
delivery to motor neurons after retrograde transport of viral par-
ticles injected intramuscularly (i.m.; Kaspar, 2003; Miller et al.,
2005). This strategy enabled the delay of disease onset and the
increase of lifespan in a mouse model of amyotrophic lateral scle-
rosis (ALS; Kaspar, 2003). However, targeting specic brain areas
such as the cerebral cortex would have required repeated injections,
thus preventing the clinical application of such an approach (Foust
et al., 2008). Conversely, efcient brain transduction after single
systemic injection of AAV particles has been recently reported in
several species such as mice, rats, cats, and monkeys (Table 1;
Foust et al., 2008; Duque et al., 2009; Wang et al., 2010; Dehay
et al., 2012). Foust et al. (2008) demonstrated a greater neuronal
tropism after injection in neonatal mice through the facial vein
while injection into adult mice through the tail vein led to glial
(mostly astrocytic) transduction. However, Duque et al. (2009)
reported up to 28% of transduction of cervical spinal cord in adult
mice after intravascular (i.v.) injection suggesting that this route
of injection might be effective for brain transduction in adult ani-
mals, even though the transduction efciency was variable. Again,
systemic injection of rAAV9 to neonate cats also showed better
transduction efciency than in adult animals (Duque et al., 2009).
Intravenous administration of rAAV9 to neonatal rats showed
up to 78% of transduction of motor neurons of the spinal cord
associated with widespread CNS transduction (Wang et al., 2010).
Altogether, these studies suggest that injection in neonatal ani-
mals is more successful compared to injection in adult animals
for widespread brain transduction. This strategy has been success-
fully applied to a spinal muscular atrophy (SMA) mouse model
(Foust et al., 2010). In this pioneer work, the group of Brian Kas-
par injected rAAV9 expressing the survival motor neuron (SMN)
protein at postnatal day 1 (P1) into SMA animals allowing rescued
motor function and increased lifespan (Foust et al., 2010). Interest-
ingly, treatment at postnatal day 5 partially rescued the phenotype
while treatment at postnatal day 10 had barely any effect (Foust
et al., 2010). The decreased effect of treatment over time can be
correlated with the increased glial transduction. To date, it is not
yet fully understood why neuronal transduction in adult brains is
not as powerful as in neonatal animals. Several factors have been
proposed such as differences in extracellular matrix composition,
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Systemic AAV-mediated gene delivery to the CNS
neuron-to-glia ratio or BBB maturity although these hypotheses
remain controversial (Lowenstein, 2009; Saunders et al., 2009).
Recently, the group of Andrea Ballabio used a combined approach
with both intracerebral ventricle injection and systemic injection
of rAAV9 to achieve whole-body transduction in a multiple sul-
fatase deciency (MSD) mouse model (Spampanato et al., 2011).
Although the combined approach reverses the phenotype of this
severe LSD, the intracerebral ventricle injection explained mainly
the brain transduction while the systemic injection induced most
of the peripheral transduction (Spampanato et al., 2011). Sev-
eral studies conrmed the reproducibility of rAAV9 intravenous
injection with a dose-dependent CNS transduction in neonatal
mice associated with a sustained expression of up to 18 months
post-injection (Gray et al., 2011b; Miyake et al., 2011). While most
studies used rAAV9, several other serotypes have also been shown
to cross the BBB and induce a robust CNS transduction as well
(Zincarelli et al., 2008; Zhang et al., 2009). Among them, rAAVrh10
appeared at least as efcient as rAAV9 in CNS transduction after
i.v. injection into neonatal mice (Zhang et al., 2009).
Consistent with mouse, rat, and cat studies, efcient gene
delivery and brain transduction has been reported after systemic
injection of rAAV in macaque monkeys (Bevan et al., 2011; Gray
et al., 2011b; Dehay et al., 2012; Mattar et al., 2012; Samaranch
et al., 2012). Even though some methodological discrepancies
between studies prevent a clear comparison of the results, some
conclusions can be achieved. Systemic administration of rAAV9 to
adult monkeys induced mostly glial transduction (Bevan et al.,
2011; Gray et al., 2011b; Samaranch et al., 2012) while injec-
tion in neonate animals induced neuronal transduction (Dehay
et al., 2012; Mattar et al., 2012). Another concern in this eld
of research involves anti-AAV antibodies, which are commonly
present in both non-human primate and humans (Boutin et al.,
2010; Calcedo et al., 2011; Gray et al., 2011b; Samaranch et al.,
2012). Such antibodies might prevent efcient brain transduction
and might explain the weaker transduction into adult animals
since anti-AAV antibody concentration has been reported to
increase with time suggesting that the sooner the better is
the credo for systemic injection in NHP (Calcedo et al., 2011).
However, with regard to these discrepancies between studies, a
high-titer dose of viral particles appeared to be the common
denominator for monkey injection (Bevan et al., 2010; Gray et al.,
2011b; Dehay et al., 2012; Mattar et al., 2012; Samaranch et al.,
2012).
These critical points in mind, gene delivery to fetuses could be
clinically relevant for early-onset diseases associated with neurode-
generation and early death in childhood such as Type II Gaucher
disease (GD). In this particular disorder, brain pathology can be
detected in utero and death occurs within the two rst years of age
(Sidransky et al., 2009). Correspondingly, two studies adminis-
tered rAAV9GFP to fetal mice or monkeys and reported a robust
central and peripheral transduction (Rahim et al., 2011; Mattar
et al., 2012). Both studies reported a strong transduction of neu-
ronal cells compared to astrocytes, surpassing that of neonatal
injection (Rahim et al., 2011; Mattar et al., 2012). Even though
this strategy has not been applied yet to a disease model such as
Type II GD, in utero delivery of a therapeutic gene might improve
phenotype of early-onset diseases.
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 Table 1 | Summary of literature reports using AAV vectors for systemic gene delivery to the CNS in mammals.

Study Serotype Virus type Reporter Administration Animal Titer Remarks

Bourdenx et al.

Miyake et al. (2011) 1, 8, 9, 10 SS GFP i.v. (jugular vein) Mice (PI, P5, P14, P42) 1.5 l011 vg/pup rAAV9 was the most efcient
1.5 1012 vg/adult Transduction efciency decrease over
time
Duque et al. (2009) 9 SC GFP i.p Mice (PI and 8 weeks) i.p: 1010 vg/mouse i.v. route of delivery was superior to the
i.m. (triceps and Cats (P2 and 7 weeks) i.m.: 109 vg/mouse other
gastrocnemius) i.v.: 1011 vg/kg

Frontiers in Molecular Neuroscience

i.v. (temporal vein)
Zhang et al. (2011) 1, 2, 5, 6, 6.2, 7, 9, SC eGFP i.v. (temporal vein) Mice (PI) 4 1011 9, rhlO, rh39, rh43 were the most
rhlO, rh39, rh43 efcient
Rahim et al. (2011) 9 SS and SC GFP in utero (vitelline Mice (E15 and PI) 2 lO11 vg/embryo In utero delivery was more efcient
vessels) i.v. (temporal 4 l011 vg/pup than PI
vein)
Mattar et al. (2012) 9 SC eGFP In utero Cynomolgus macaques 3 1011 vg/kg Transduction was mostly neuronal, up
(E140) to 97% in the cerebellum
Zincarelli et al. (2008) 1, 2, 3, 4, 5, 6, 7, SS Luciferase i.v. (tail vein) Mice (810 weeks) 1 1011 Luciferase activity in the brain was
8, 9 detected only after rAAV8 or -9 injection
Dehay et al. (2012) 9 SC GFP i.v. (saphenous vein) Rhesus macaques 1.33 1015 vg/kg Post-natal developed structures were
more transduced than the others
Bevan et al. (2011) 9 SC GFP i.v. (saphenous vein) Cynomolgus Macaques Injection to young animal was more

www.frontiersin.org
l3 l014 vg/kg to
(P1P90, 3-year-old) PlP90 animals powerful compared to injection to adult
2.7 1011 vg/kg to adult (partially attributable to the dose)
animals
Samaranch et al. (2012) 9 SS and SC GFP i.v. (carotid artery) Cynomolgus and Rhesus 3 1013 vg/kg for i.v. Similar pattern for both route
i.cm. macaques 1.8 l03 vg/kg for i.c.m. injection led to stronger
expression than i.v. injection
Gray et al. (2011b) 9 SS and SC GFP Mice: i.v. (tail vein) Mice (812 weeks old) Mice: up to Dose-dependent transgene expression
Monkey: i.v. Rhesus monkeys 8 1013 vg/kg Shift toward glial transduction was
(saphenous vein) (34-year-old) Monkey: l 1011 vg/kg more pronounced in monkeys
Foust et al. (2008) 9 SC GFP Neonates: i.v. (facial Mice (PI and 10 weeks) 4 1011 vg/animal Neuronal transduction in neonates
vein) Astrocytic transduction in adults
Adults: i.v. (tail vein)

SS, single stranded; SC, self-complementary; GFP, green uorescent protein; eGFP, enhanced GFP; i.v., intravascular; i.m., intramuscular; i.p, intraperitoneal; i.c.m., intra cisterna magna; P1, postnatal day 1; E15,
embryonic day 15; vg, virus genome.

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Systemic AAV-mediated gene delivery to the CNS
Bourdenx et al.
Several routes of administration have been tested to obtain
widespread brain transduction. As mentioned earlier, large brain
transduction has been reported after intravenous injections of
rAAV (Foust et al., 2010; Dehay et al., 2012). Interestingly, a single
intracardiac injection of rAAV to adult mice has been reported to
preferentially transduce glial cells and to reduce amyloid- peptide
levels in the brain (Iida et al., 2013; Iwata et al., 2013). Once again,
the strong glial transduction might be due to the time of injec-
tion. More invasive protocols such as intrathecal intra-cisterna
magna injections of rAAV9 have been reported to efciently trans-
duce cerebral tissue in both NHPs and pigs (Federici et al., 2012;
Samaranch et al., 2012). Intra-cisterna magna and other intrathe-
cal injections reported a stronger transduction compared to i.v.,
i.m., s.c., and sciatic nerve injections (Towne et al., 2009; Sama-
ranch et al., 2012). Although interesting, this approach could
not circumvent the antibodies issue in NHP (and hence man;
Samaranch et al., 2012). Finally, some groups used intranasal
administration of rAAV and reported an efcient transduction
mostly in olfactory bulbs and lungs (Zhang et al., 2003; Wolf et al.,
2012).
Widespread brain transduction after systemic injection of rAAV
might have two distinct applications. First, AAV-mediated expres-
sion of a given pathogenic protein in the whole brain could
be an alternative modeling system to the classic toxic-based
or local injection models of non-focal neurological disorders.
Moreover, such strategy might allow global CNS transduction
of animals such as rats or NHP, which remains difcult to
achieve by classical transgenesis. Second, widespread silencing
of a pathogenic protein or expression of a rescue protein might
be useful for therapeutic interventions. However, gene therapy
for neurodegenerative disorders, which are mainly adult-onset
diseases, would ideally occur in adult, unless familial diseases
or cases of otherwise sporadic diseases are targeted. Even if
glial transduction might have clinical relevance for diseases such
as amyotrophic lateral sclerosis (ALS) or Parkinsons disease
(Nagai et al., 2007; Colangelo et al., 2014), a strong cell-type-
specic transduction is mandatory for clinical application of
systemic gene delivery via rAAV. Moreover, the large titers of
virus used in NHPs suggest that even higher titers should be
used in humans. These limitations stress the need for more
powerful and precise vectors as well as an optimized route of
administration.
CONTROL OF TRANSGENE EXPRESSION, CELL SPECIFICITY,
AND VECTOR OPTIMIZATION
VIRUS GENOME TUNING
To overcome these limitations, several methods have been devel-
oped to improve brain transduction after systemic injection
(Figure 1). The WT AAV genome is packaged as a linear ssDNA
with ITRs at both ends. Host-cell-mediated synthesis of the
second strand of the AAV genome has been shown to be the
rate-limiting step of transduction with rAAV (Ferrari et al., 1996).
Thus, McCarty et al. (2003) deleted the terminal resolution site
from one ITR to generate so-called self-complementary vectors
(scAAV) with a 10 to 50-fold stronger gene expression than single-
stranded vectors. However, such an increase in gene expression
leads to the loss of half of the packaging capacity of the vector
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Systemic AAV-mediated gene delivery to the CNS
(2150 bp for scAAV2; McCarty et al., 2003). Such increased expres-
sion has been reported in a systemic gene delivery study where the
number of reporter-positive cells after ssAAV9 injection was sim-
ilar to that obtained with a 20-fold lower dose of scAAV9 (Gray
et al., 2011b).
One of the main concerns of systemic gene delivery via i.v.
injection of rAAV might be off targets. Several studies reported
transduction of peripheral organs, such as skeletal muscle, heart,
pancreas, or antigen-presenting cells after systemic injections
(Zincarelli et al., 2008; Rahim et al., 2011; Mattar et al., 2012).
Although it can be of signicance for diseases with periph-
eral and central components such as LSD, this can raise the
apprehension of unwanted protein overexpression external to
the CNS potentially eliciting toxic responses (Xie et al., 2011).
At the vector genome level, two distinct but complimentary
strategies could be used to specify gene expression: the rst
is based on cell-type-specic promoters restricting transgene
expression to certain cell subpopulations; conversely the sec-
ond involves the repression of transgene expression in unwanted
cells or organs. In this context, promoter choice is critical
since it can determine the strength or specicity of expression.
Most intracerebral AAV injections used a cell-type-specic pro-
moter such as the synapsin promoter to restrict expression to
neurons (Decressac et al., 2012; Engeln et al., 2013). However,
systemic gene delivery studies mostly use strong and ubiqui-
tous promoters including the cytomegalovirus (CMV) promoter
or the truncated chicken beta actin (CBA) promoter (Dalkara
et al., 2011; Mattar et al., 2012). The restricted packaging capac-
ity of ssAAV and even more of self-complementary vectors
stresses the need for minimal and strong promoters. To fulll
this need, Gray et al. (2011a) developed a hybrid CBA (CBh)
promoter of 800 base pairs (bp) allowing more stable, longer,
and stronger expression compared to CMV or CBA promoters.
This expression can be further enhanced by the use of 5 or
3 untranslated regions (UTR). Of note, the woodchuck hep-
atitis virus post-transcriptional response element (WPRE) has
been shown to improve brain transduction after intracerebral
injection (Hermening et al., 2006; Decressac et al., 2012). This
increased transduction comes with a cost of 600 bp of packag-
ing size (Hermening et al., 2006). Rahim et al. (2011) compared
brain and eye expression after in utero injection of scAAV9 with-
out WPRE element versus ssAAV9 carrying the WPRE sequence.
They observed that the scAAV9 vector including the WPRE
sequence is more efcient than an scAAV9 vector deprived of
WPRE. Subpopulation-specic neuronal promoters are often long
DNA sequences with regulatory elements. For instance, the full
mouse tyrosine hydroxylase promoter that controls expression
in dopaminergic neurons is 7.5 kb making it too voluminous
for use in an rAAV (Iwata et al., 1992). In this context, the use
of tissue-specic microRNAs (miRNAs)-binding site in the AAV
genome could overcome this promoter size limitation by repress-
ing expression in tissue that express the miRNAs (Xie et al., 2011).
Such a strategy might be of interest for systemic gene delivery.
Indeed, the incorporation of three copies of miRNA122-binding
site or miRNA1-binding site in the rAAV genome dramatically
decreases transduction in liver or heart, respectively (Xie et al.,
2011). Reduction of hepatic transgene expression after systemic
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Bourdenx et al.
FIGURE 1 | Summary of methods used to improve gene delivery after systemic injection of AAVs. AAV, adeno-associated virus; BBB, bloodbrain barrier;
miRNA, microRNA; VP, viral particle protein.
gene delivery might be an added benet as liver is a key target of
AAV vectors.
CAPSID TUNING
Virus capsid is the other obvious target to engineer to improve
or specify transgene expression. A WT AAV capsid comprises
three structural Cap proteins: VP-1, -2, and -3 with a ratio of
1:1:10. The capsid is the primary interface between virus and
host cell, mediating vector binding to cell surface receptors (Wu
et al., 2006). Moreover, the capsid but not the ITR sequence inu-
ences cell and tissue tropism (Grimm et al., 2008; Vandenberghe
et al., 2008). The propensity of certain AAVs serotypes to bypass
anatomical barriers is directly related to their capsid composi-
tion since only several serotypes with identical genome are able to
efciently cross the BBB (Zhang et al., 2011). Glycans with ter-
minal -galactose linkages have been recently identied as the
primary receptor for AAV9 (Bell et al., 2011; Shen et al., 2011).
This unique feature of serotype 9 might be related to its ability to
cross the BBB. The 37/67-kDa laminin receptor has been identied
as co-receptor for AAVs of serotypes 2, 3, 8, and 9; however, its
involvement in BBB crossing has not been determined yet (Akache
et al., 2006).
Capsid engineering can be designed to produce new AAV
variants by (i) mutagenesis of VP proteins, (ii) incorporation
of specic peptide ligand at the virus surface or (iii) directed
evolution (Gray and Samulski, 2011). One example of cap-
sid mutagenesis is tyrosine substitution. Zhong and colleagues
reported that rAAV2 capsid could be phosphorylated on surface-
exposed tyrosines leading to ubiquitinylation and degradation of
viral particles (Zhong et al., 2008). Mutagenesis of one or more of
the seven surface-exposed tyrosine residues to phenylalanine (Y
F) has been reported to reduce proteasomal degradation of viral
particles and therefore enhance retina transduction after either
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Systemic AAV-mediated gene delivery to the CNS
systemic or intravitreous injection of rAAV2, -8, or -9 (Petrs-Silva
et al., 2008; Zhong et al., 2008; Dalkara et al., 2011). Similarly, the
group of Aravind Asokan used random mutagenesis to identify
two rAAV9 variants carrying one (N498I) or two (N498Y and
L602F) mutations associated with a 10-fold decreased liver trans-
duction without affecting transduction of other organs after a
tail vein injection (Pulicherla et al., 2011). Earlier work reported
that rAAV2 capsid could sustain insertional mutagenesis with-
out affecting infectivity (Rabinowitz et al., 1999). In this context,
peptides derived from a glutamatergic receptor antagonist and
dynein-binding motif have been inserted in the VP3 sequence
allowing delivery and retrograde transport of rAAV2 to the CNS
after peripheral (tongue) injection in vivo (Xu et al., 2005). Several
groups inserted peptide motifs into rAAV2 capsid after random
library or phage-display library screening to increase rAAV2 afn-
ity for coronary or cerebral endothelium (Mller et al., 2003; Chen
et al., 2009). Although very promising, further work is neces-
sary to characterize such peptides for brain transduction. DNA
shufing and directed evolution are other methods used to gen-
erate mixtures of AAV capsid genes in an unbiased way. Several
methods have now been described, but they are all based on a
two-step strategy (Maheshri et al., 2006; Gray et al., 2010; Dalkara
et al., 2013). First, a library is created by error-prone polymerase
chain reaction to induce random mutagenesis on capsid genes
or by a combination of different serotypes or random insertion
libraries. Second, the library is subjected to several rounds (often
three) of selection. One or more clones are obtained with unique
characteristics. The Samulskis group used this method to cre-
ate clones that can selectively cross the seizure-compromised BBB
and transduce specic cells at the damage sites without transduc-
ing other organs (Gray et al., 2010). The new vectors generated
by directed evolution or a similar strategy might improve neu-
ronal transduction in adult and also overcome the seropositivity
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Bourdenx et al.
problem, since no antibody can be generated for these new capsid
variants.
Several methods, not directly relying on the virus, have been
proposed to increase brain expression after systemic delivery of
rAAV to adult animals. Mannitol has been used to induce hyper-
osmotic breaching of the BBB, therefore increasing the entry of
AAV into the brain (Fu et al., 2011). Although positive results were
reported for rAAV2 in a mouse model of LSD, co-administration of
mannitol with rAAV9 had only modest effects on brain transduc-
tion (McCarty et al., 2009; Fu et al., 2011; Gray et al., 2011b). Those
studies suggest that rAAV9 crosses the BBB through active trans-
port (Gray et al., 2011b). Moreover, mannitol co-administration
could be risky since it increases the inux of all molecules in the
brain. However, this compound is regularly used in clinical prac-
tice and no adverse effects have ever been reported in clinical trials
(Kaplitt et al., 2007; Lowenstein, 2009). Identication and mod-
ication of the key components of such transport might allow
transduction improvements. As stated earlier, preexisting anti-
AAV antibodies in primate might represent a major obstacle to
AAV-mediated gene therapy success. To overcome this concern,
Katherine A. Highs group developed an empty mutant capsid,
which can interact with antibodies without entering the cell (Min-
gozzi et al., 2013). As a trojan, addition of the mutant capsid at
different ratios in the whole vector formulation increased trans-
duction with the same vector genome dose in both mouse and
NHPs (Mingozzi et al., 2013). While safety and efcacy have been
proved, this encouraging concept still needs to be tested in a disease
model.
CONCLUDING REMARKS
Ultimately, gene therapy has been a long sought goal for neuro-
logical disorder. Thirty-two years after the development of the
rst recombinant virus, AAV appears to be an extremely use-
ful and promising lynchpin for both therapeutic approaches to
neurodegenerative disorders and useful strategies in neuroscien-
tic research. Recent ndings demonstrated that several serotypes,
such as AAV9 or AAVrh10, cross the BBB with a safe systemic deliv-
ery protocol associated with strong non-focal brain transduction.
Such a strategy has been proved efcient in several animal mod-
els of disease such as SMA or LSDs. Moreover, several innovative
strategies, at the genome or capsid levels, have been developed to
increase and/or precise tissue-specic gene expression after sys-
temic injection of rAAV. Unavoidable efforts need to be done
to harmonize production, purication, titration, and injection
protocols between laboratories. It is worth noting that strength-
ening those specic points will help achieve a clear comparison
between these studies. The advancements in AAV-mediated gene
therapy hold the promise of a bright future for neurodegenerative
diseases.
ACKNOWLEDGMENTS
The authors thank Cynthia Lebeaupin for valuable comments
on the manuscript. The Universit of Bordeaux and the Cen-
tre National de la Recherche Scientique provided infrastructural
support. This work was supported by Marie Curie Reintegration
Grant FP7-PEOPLE-2009-ERG256303 from the European Com-
mission (to Benjamin Dehay); by a grant from the Fondation pour
Frontiers in Molecular Neuroscience www.frontiersin.org
Systemic AAV-mediated gene delivery to the CNS
la Recherche Mdicale (to Benjamin Dehay); by Agence Nationale
de la Recherche Grants ANR-08-MNP-018 and ANR-07-MNP-
Tranlid (to Erwan Bezard) and Mathieu Bourdenx is a recipient
of an MESR fellowship.
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or nancial relationships that could be construed
as a potential conict of interest.
Received: 03 March 2014; paper pending published: 28 April 2014; accepted: 14 May
2014; published online: 02 June 2014.
Citation: Bourdenx M, Dutheil N, Bezard E and Dehay B (2014) Systemic gene delivery
to the central nervous system using Adeno-associated virus. Front. Mol. Neurosci. 7:50.
doi: 10.3389/fnmol.2014.00050
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